ALBERT

Description: Identification of growth factors in neoplasias may be a target for future therapies by blocking either growth factor receptor interaction or the induced pathway. Using gene expression profiling, we identified overexpression of 2 receptors for a proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) in malignant plasma cells compared with normal plasma cells. APRIL and BAFF are involved in a variety of tumor and autoimmune diseases, including B-cell malignancies. We confirmed the expression of BAFF and APRIL receptors (B-cell maturation antigen [BCMA], transmembrane activator and calcium modulator and cyclophilin ligand interactor [TACI], and BAFF-R) in a majority of 13 myeloma cell lines and in the purified primary myeloma cells of 11 patients. APRIL and BAFF were potent survival factors for exogenous cytokine-dependent myeloma cell lines and were autocrine growth factors for the RPMI8226 and L363 autonomously growing cell lines. These factors activated nuclear factor (NF)–κB, phosphatidylinositol-3 (PI-3) kinase/AKT, and mitogen-activated protein kinase (MAPK) kinase pathways and induced a strong up-regulation of the Mcl-1 and Bcl-2 antiapoptotic proteins in myeloma cells. BAFF or APRIL was also involved in the survival of primary myeloma cells cultured with their bone-marrow environment, and protected them from dexamethasone (DEX)–induced apoptosis. Finally, the serum levels of BAFF and APRIL were increased about 5-fold in patients with multiple myeloma (MM) as compared with healthy donors. Altogether, these data suggest that APRIL/BAFF inhibitors may be of clinical value in MM. (Blood. 2004;103:3148-3157)

Description: Multiple myeloma is characterized by the clonal expansion of malignant plasma cells (multiple myeloma cells [MMCs]), in the bone marrow. Osteolytic bone lesions are detected in 80% of patients because of increased osteoclastic bone resorption and reduced osteoblastic bone formation. MMCs are found closely associated with sites of increased bone resorption. Osteoclasts strongly support MMC survival in vitro. To further elucidate the mechanisms involved in osteoclast/MMC interaction, we have identified 552 genes overexpressed in osteoclasts compared with other bone marrow cell subpopulations. Osteoclasts express specifically genes coding for 4 CCR2-targeting chemokines and genes coding for MMC growth factors. An anti-CCR2 monoclonal antibody blocked osteoclast chemoattractant activity for MMC, and CCR2 chemokines are also MMC growth factors, promoting mitogen-activated protein kinase activation in MMC. An anti-insulin growth factor-1 receptor monoclonal antibody completely blocked the osteoclast-induced survival of MMC suppressing both osteoclast and MMC survival. Specific a proliferation-inducing ligand or IL-6 inhibitors partially blocked osteoclast-induced MMC survival. These data may explain why newly diagnosed patients whose MMC express high levels of CCR2 present numerous bone lesions. This study displays additional mechanisms involved in osteoclast/MMC interaction and suggests using CCR2 and/or insulin growth factor-1 targeting strategies to block this interaction and prevent drug resistance.

Description: B-cell activating factor (BAFF) and A Proliferation-Inducing Ligand (APRIL) are produced by the bone marrow microenvironment of patients with multiple myeloma (MM) and are growth factors of multiple myeloma cells (MMC). BAFF and APRIL share two receptors - TACI and BCMA - and BAFF binds to a third receptor, BAFF-R. We previously reported that TACI expression is a good indicator of a BAFF-binding receptor in human myeloma cell lines (HMCL), unlike BCMA that is expressed on all HMCL. BAFF-R is lacking. More recently, Ingold et al (J Exp Med; 2005) and Hendriks at al (Cell Death Differ; 2005) identified proteoglycans as the APRIL-specific binding partners. Bischof et al (Blood; 2006) demonstrated that TACI binds also heparan sulfate (HS) chains, in particular to syndecan-1, syndecan-2 and syndecan-4. Syndecan-1 is expressed by plasma cells and epithelial cells and is involved in several cellular processes relying on interactions with extra-cellular matrix proteins, growth factors, chemokines and adhesion molecules. We found a very large binding of APRIL (mean fluorescence activity ≥ 500), unlike BAFF, at the surface of all syndecan-1+ HMCL. These syndecan-1+ HMCL also highly bound TACI-Fc molecules, unlike BCMA-Fc or BAFF-R-Fc molecules. This large binding of APRIL or TACI-Fc molecules was abrogated by heparitinase pretreatment of MMC, removing the HS chains or by preincubation of APRIL and TACI-Fc with heparin. These data were extended to patients primary myeloma cells. In agreement with this large binding to MMC, APRIL was 15 to 40 fold more efficient than BAFF to stimulate the growth of HMCL. The APRIL growth factor activity could be inhibited using heparin, unlike that of BAFF. Our data establish that syndecan-1, by concentrating large levels of APRIL and TACI at MMC surface, can promote APRIL/TACI signaling that induces survival and proliferation of MMC. Heparan sulfate chain inhibitors could be helpful to synergize with BAFF/APRIL inhibitors in MM disease.

Description: Using Affymetrix microarrays, we identified the expression of the CD200 gene in multiple myeloma cells (MMC) of 112 patients with newly-diagnosed multiple myeloma (MM). The CD200 gene was either absent or present (Affymetrix call) in 22% and 78% of MMC, respectively. CD200 was not expressed by CD14 monocytes, CD15 polynuclear cells and CD3 T cells that were purified from the bone marrow of 5 newly-diagnosed patients. It is also not expressed in 7 osteoclast samples. BM stromal cells from 5 patients with MM expressed CD200, but at a 3.9 fold lower median signal compared to that in CD200present MMC (P = .04). CD200 is a membrane glycoprotein that imparts an immunoregulatory signal through CD200R, leading to the suppression of T-cell-mediated immune responses. Patients with CD200absent MMC have an increased event free survival (24 months) compared to patients with CD200present MMC (14 months), after high-dose therapy and stem cell transplantation. In a Cox-proportional-hazard model, the absence or presence of CD200 expression in MMC is predictive for EFS for patients independently of ISS stage or B2M serum levels. Thus, CD200 is an independent prognosis factor for patients with MM that could represent a new therapeutic target in MM.

Description: Multiple myeloma (MM) is a fatal hematologic malignancy associated with clonal expansion of malignant plasma cells within the bone marrow and the development of a destructive osteolytic bone disease. The principal cellular mechanisms involved in the development of myeloma bone disease are an increase in osteoclastic bone resorption, and a reduction in bone formation. Myeloma cells (MMC) are found in close association with sites of active bone resorption, and the interactions between myeloma cells and other cells within the specialized bone marrow microenvironment are essential, both for tumor growth and the development of myeloma bone disease. In order to investigate the gene expression profile (GEP) of osteoclastic cells, we compare GEP of osteoclastic cells (7 samples) with normal B cells (7 samples), normal bone marrow plasma cells (7 samples), bone marrow stromal cells (5 samples), bone marrow CD3 cells (5 samples), CD14 cells (7 samples), CD15 cells (7 samples), CD34 cells (7 samples) and primary MMC (123 samples). Using SAM analysis, a set of 552 genes was overexpressed in osteoclasts compared to others cell subpopulations with a FDR ≤ 1% and a ratio ≥ 2. Osteoclasts specifically overexpressed genes coding for chemokines (CCL2, CCL7, CCL8, CCL13, CCL18, CXCL5 and CCL23) and MMC growth factors (IGF-1, APRIL and IL-10). Anti- IGF-1 receptor and TACI-Fc inhibit MMC growth induced by osteoclasts. Among the chemokines overexpressed by osteoclasts, the majority of them have a common receptor: CCR2 expressed by MMC. Anti-CCR2 MoAb inhibits migration of the CCR2+ HMCL in response to osteoclasts. Expression data of purified MMC were analyzed by supervised clustering of group with higher (CCR2high) versus lower (CCR2low) CCR2 expression level. Patients in the CCR2high group are characterized by a higher bone disease. A set of 176 genes was differentially expressed between CCR2high and CCR2low MMC. CCR2high displayed a gene signature linked to the dependency of MMC on the interactions with the BM osteoclastic subpopulation and the osteoclastic bone resorption. Taken together, our findings suggest addition of chemokine antagonists to current treatment regimens for MM should result in better therapeutic responses because of the loss of both the protective effect of the bone marrow environment on the MMC and the osteoclastic cells activity.